Food Chemistry 135 (2012) 1173–1182 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Picea mariana bark: A new source of trans-resveratrol and other bioactive polyphenols Martha-Estrella García-Pérez a,b,c, Mariana Royer a, Gaëtan Herbette d, Yves Desjardins e, Roxane Pouliot b,c, Tatjana Stevanovic a,⇑ a Centre de Recherche sur le Bois, Département des sciences du bois et de la forêt, Faculté de foresterie et géomatique, Université Laval, Québec, QC, Canada G1V 0A6 Centre LOEX de l’Université Laval, Génie tissulaire et régénération, LOEX – Centre de recherche FRSQ du Centre hospitalier affilié universitaire de Québec, Aile-R, 1401 18e rue, Québec, QC, Canada G1J 1Z4 c Faculté de Pharmacie, Université Laval, Québec, Qc, Canada G1V 0A6 d Spectropole, FR 1739-Aix-Marseille Université, Campus de Saint-Jérome Service 511, 13397 Marseille, Cedex 20, France e Institut des Nutraceutiques et des aliments fonctionnels, Centre de recherche en Horticulture, Pavillon de l’Envirotron, 2480 Boul. Hochelaga, Université Laval, Québec, QC, Canada G1V 0A6 b a r t i c l e i n f o Article history: Received 2 March 2012 Received in revised form 7 May 2012 Accepted 10 May 2012 Available online 22 May 2012 Keywords: Bark Black spruce Picea mariana Lignans Neolignans Polyphenols Resveratrol a b s t r a c t The ethyl acetate soluble fraction obtained from the hot water extract of Picea mariana bark (BS-EAcf) has been demonstrated to have anti-inflammatory and antioxidant properties. Thus, in the current study, we isolated and characterised major compounds of this fraction by HPLC, NMR and MS analyses. On the whole, 28 compounds were identified, among which were five neolignans, seven lignans, transresveratrol, three phenolic acids and four flavonoids. To the best of our knowledge, 2,3-dihydro-3-(4hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-(2S,3S)-1,4-benzodioxin-6-propanol, threo and erythro 3-methoxy-8,40 -oxyneolignan-30 ,4,7,9,90 -pentol, pallasiin, (±) epi-taxifolin, homovanillyl alcohol, orcinol and 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol are reported for the first time in the Picea genus. P. mariana dry bark contains at least 104 lg g 1 dw of trans-resveratrol and it could be therefore considered as a new accessible source of this molecule. This study provides novel information about the identity of major compounds present in BS-EAcf, which is essential for the understanding of the anti-inflammatory and nutraceutical potential of this extract. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Polyphenols are ubiquitous plant constituents that exhibit a wide range of physiological effects, acting as antioxidant, antiallergenic, anti-inflammatory, anticarcinogenic and cardioprotective agents (Chandrasekara & Shahidi, 2011; Kang, Shin, Lee, & Lee, 2011; Oh et al., 2009). Bioactive polyphenols, partially responsible for the health benefits of diets rich in fruits and vegetables, are also available from forest trees and in particular from the residues of industrial wood transformation, such as barks (Stevanovic, Diouf, & García-Pérez, 2009). In Canada, large amounts of bark are produced as residues of wood transformation, being mainly burnt in large furnaces to satisfy at least a part of the forest industry’s energy needs (Diouf, Stevanovic, & Cloutier, 2009). Bark is generally considered a richer source of bioactive polyphenols than other tree organs due in part to its protective role (Gao, Shupe, Eberhardt, & Hse, ⇑ Corresponding author. Address: Centre de Recherche sur le Bois, Université Laval, Département des sciences du bois et de la forêt, Pavillon Gene H. Kruger, 2425 rue de la Terrasse, QC, Canada G1V 0A6. Tel.: +1 418 656 2131x7337; fax: +1 418 656 209. E-mail address: Tatjana.Stevanovic@sbf.ulaval.ca (T. Stevanovic). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.050 2007). This residue is thus most likely a rich source of nutraceutical supplements, dietary additives and/or pharmaceutical products. Black spruce (Picea mariana (Miller) B.S.P) is one of the most important industrial species from Canadian forests and its barks are available in huge quantities as a result of wood transformation. Recently our research group has demonstrated that the hot water extract from black spruce bark (BSHWE) presented potential as a natural nutraceutical for applications in which antioxidant and anti-inflammatory properties are important (Diouf et al., 2009). In a further investigation, it was also demonstrated that this extract possessed a high content of total phenols and flavonoids as well as a low toxicity on normal human keratinocytes and an adequate chemical reactivity towards different free radicals involved in psoriasis, a skin disorder affecting up to 2% of the world’s population (Garcia-Perez et al., 2010). For these reasons, it was considered a valuable source of bioactive molecules. Considering that BSHWE was a crude extract composed of many compounds, we have decided to fractionate it further in order to enhance its biological activity and to identify and characterise in more depth its major molecules (Garcia-Perez et al., 2010). Previous investigations also performed by our research group have shown that the ethyl acetate fraction isolated from this 1174 M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 extract (BS-EAcf) has a higher antiradical efficiency and a better antiinflammatory activity than that of BSHWE (Diouf, Stevanovic, & Boutin, 2009). Moreover, this fraction at 500 lg mL 1 (a non-toxic concentration) suppressed interleukin (IL) IL-8 production by normal and psoriatic keratinocytes stimulated with tumour necrosis factor (TNF-a) cytokine (García-Pérez et al., 2011). On the whole, these results suggest that molecules present in the BS-EAcf have potential anti-inflammatory and antioxidant properties and could be used as a part of nutraceutical or pharmaceutical products. The thiolysis coupled with HPLC-DAD used in our previous study allowed the characterisation of proanthocyanidins in this fraction (Diouf et al., 2009). However, major low molecular weight polyphenols present in BS-EAcf remains to be identified. Indeed, no comprehensive studies are presently available concerning the characterisation of low molecular weight polyphenols in P. mariana bark. An early study reported the presence of 2.5% leucoanthocyanidins in the black spruce internal bark (Pigman, Anderson, Fischer, Buchanan, & Browning, 1953). In 1971, Manners and Shawn described the occurrence of taxifolin, catechin, epicatechin and some stilbenes, such as astringin, isorhapontin, isorhapontigenin and astringenin, in the acetone extract of black spruce bark (Manners & Swan, 1971). Recently, the flavonoid taxifolin was also identified by our group both in BSHWE (13.4 mg g 1 extract) and in BS-EAcf (66.7 mg g 1 extract) (Diouf et al., 2009). Taking into account the potential of molecules present in BSEAcf as anti-inflammatory and antioxidant agents, and the fact that very few reports exist to date on the characterisation of polyphenolic compounds in P. mariana bark, this study aimed to isolate, characterise and quantify the individual compounds present in the ethyl acetate fraction of crude aqueous extract. Considering the high number of isomers and the diversity of molecules present in black spruce bark, the extraction and purification techniques here reported could be used for the production of sufficient quantities of pure compounds to study their potential as antioxidant and anti-inflammatory agents. 2. Materials and methods 2.1. Reagents Folin–Ciocalteu phenol reagent, gallic acid, quercetin and chlorogenic acid were purchased from Sigma–Aldrich (St. Louis, MO). Ammonium sulphate from Laboratoire MAT (Québec, QC, Canada) and cyanidin chloride from Indofine Chemical Co (Hillsborough, NJ) were also used. Hexane, acetonitrile, dichloromethane and ethyl acetate were obtained from Fisher Scientific Chemicals (Tustin, Canada). 2.2. General experimental procedures Contents of the various classes of polyphenols present in BS-EAcf were determined using a UV–visible spectrometer (Varian model Cary 50). Isolation of pure compounds was performed on a semi-preparative column (Zorbax SB-C18, 5 lm, 9.4  250 mm i.d., Agilent) by high-performance liquid chromatography (HPLC), along with a Agilent 1100 series system equipped with a G1311A quaternary pump, G1315B photodiode array absorbance detector and a G1364C automatic fraction collector. The samples were injected automatically through an automatic injector (900 lL maximum injection volume) and the flow rate was 4 mL min 1 for separation. Analyses of purity were performed on the same system on an analytical column (Zorbax SB-C18, 5 lm, 4.6  250 mm i.d., Agilent) with a flow rate of 1 mL min 1. Silica gel (10–40 lm, blinder CaSO4 Type G) was purchased from Sigma Aldrich and analytical TLC plates (Si gel 60 F254 20  20 mm) were purchased from EMD Chemicals Inc. After fractionation by silica gel column chromatography all fractions were dried by evaporation under vacuum, in order to determine their respective masses. The 1H and 13C NMR spectra were recorded on a Bruker Avance DRX500 spectrometer (1H-500.13 MHz) equipped with a 5-mm triple resonance inverse Cryoprobe TXI (1H–13C–15N) with a z gradient. Spectra were recorded with 1.7 mm NMR capillary tube in 40 ll of 99.99% CD3OD solvent (d1H 3.31 ppm–d13C 49.00 ppm) at 300 K. The 1H (500 MHz) and 13C NMR (125 MHz) data are reported in ppm downfield from tetramethylsilane. Hydrogen connectivity (C, CH, CH2, and CH3) information was obtained from edited HSQC and/or DEPTQ-135 experiments. Proton and carbon peak assignments were based on 2D NMR analyses (COSY, NOESY, HSQC and HMBC). HREI-MS was performed using a QStar Elite mass spectrometer (Applied Biosystems SCIEX, Concord, ON, Canada) equipped with an ESI source operating in the positive ion mode. The capillary voltage was set to 5500 V, the cone voltage to 20 V and air was used as the nebulising gas (20 psi). In this hybrid instrument, ions were measured using an orthogonal acceleration time-of-flight (oa-TOF) mass analyser. Analyst software version 2.1 was used for instrument control, data acquisition and data processing. Accurate mass measurements were performed in triplicate with two internal calibrations. Direct sample introduction was performed at a 5 lL min 1 flow rate using a syringe pump. The UV spectra were recorded on a Perkin-Elmer Lambda 5 spectrophotometer. Optical rotations were measured with a Perkin-Elmer 241 polarimeter equipped with a sodium lamp (589 nm) and a 1 dm cell. 2.3. Plant material Barks of black spruce (P. mariana (Miller) B.S.P) were from St.Lambert de Lauzon, Chaudière-Appalaches region, Québec, Canada and were identified by Alain Cloutier Ph.D., professor of wood anatomy at Université Laval. A voucher specimen was deposited in the herbarium Louis-Marie of Université Laval, Québec, QC, Canada with reference number: QFA 0579234. 2.4. Extraction and separation of the ethyl acetate fraction Hot water crude extract from black spruce bark (BSHWE) was obtained as described in our previous work (Garcia-Perez et al., 2010). Briefly, fifty grams of oven dry ground (40–60 mesh) black spruce bark were first extracted with 500 mL water under reflux for 1 h and solids were separated by filtration with a Whatman No. 4 filter paper and washed with 500 mL of hot water. The aqueous filtrate (1 L) was freeze-dried to yield BSHWE 4.98 g (9.96 ± 0.08%). The ethyl acetate fraction was separated from BSHWE as previously described (Diouf et al., 2009). Briefly, 3.25 g of BSHWE were resuspended in 100 mL water, decanted through a 100-mL Gooch crucible (PyrexÒ, 40–60 lm, coarse porosity) and the filtrate was collected in a 125-mL Erlenmeyer flask. The aqueous solution was first defatted with hexane (5  100 mL) and then extracted with ethyl acetate (5  100 mL). The organic fraction was solvent-evaporated under vacuum to remove ethyl acetate, resuspended in water, filtered through a 50-mL Gooch crucible (PyrexÒ, 40–60 lm, coarse porosity) and then lyophilised to yield the 676 mg ethyl acetate fraction (BS-EAcf) (20.8 ± 2.34%). 2.5. Polyphenols classes present in the ethyl acetate fraction of black spruce bark aqueous extract Different polyphenols classes (flavonoids, hydroxycinnamic acids and proanthocyanidins) and total phenols in the ethyl acetate fraction (BS-EAcf) were quantified by spectrophotometric methods. The total phenol content was determined using the Folin–Ciocalteu method as previously described (Diouf et al., 2009) and the results M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry extract (mg GAE g 1 dry BS-EAcf). The total flavonoid content of the BS-EAcf was determined following the method described by Lamaison and Carnat (Brighente, Dias, Verdi, & Pizzolatti, 2007) and results were expressed as milligrams of quercetin equivalents (QE) per gram of dry extract (mg QE g 1of dry BS-EAcf). The total hydroxycinnamic acid content was determined as described in European Pharmacopoeia for Fraxini folium (European Pharmacopoeia. Fraxini folium., 2002) and results were expressed as milligrams of chlorogenic acid equivalents (ChAE) per gram of dry extract (mg ChAE g 1 of dry BS-EAcf). Anthocyanidin monomer formation in a hydrochloric medium with ferric ammonium sulphate as a catalyst was performed as described by Porter, Hrstich, and Chan (1986). The proanthocyanidin content was expressed as milligrams of cyanidin chloride equivalents (CChE) per gram of dry extract (mg CChE g 1 of dry BS-EAcf). Taking into account that in this study the provenance of bark used for obtaining BS-EAcf, the extraction approach and the methods used for determining different classes of phenols were the same as those described in our previous work for BSHWE (Garcia-Perez et al., 2010), the results obtained for the extracts (BSHWE and BS-EAcf) from the two studies were compared using the Student’s t test (p < 0.05). Results were processed by SAS program 8.2 software (SAS Institute Inc., Cary, NC, USA). 2.6. Isolation of individual compounds present in the ethyl acetate fraction Fig. 1 shows the flow chart used for the isolation of individual compounds present in the ethyl acetate fraction. The dried powdered BS-EAcf (676.0 mg) was suspended in 67.6 mL water (V1) (10 mg mL 1) and sequentially partitioned with dichloromethane (phase A, 2  V1) and ethyl acetate (phase B, 3  V1 mL). Fig. 2 shows the typical RP-HPLC chromatogram of phase A (153 mg) which is composed of a complex mix of different molecules. Phase A was primarily fractionated by silica gel column chromatography (40–63 lm; 2  50 cm) with a hexane–ethyl acetate gradient as follows: 70:30; 60:40; 50:50; 40:60; 30:70 (v:v). Consequently, fifteen fractions were obtained and numbered A00-A14. Fractions A00-A08; A10 and A14 were purified by HPLC. Taking into account that fractions A09, A11, A12 and A13 presented a low mass (<1 mg) and contained several compounds as observed by TLC, they were not further sub-fractionated. Fractions A00 and A01 were purified by HPLC using a water–acetonitrile linear gradient as follows: 70:30 to 100% acetonitrile over 15 min and then 100% acetonitrile for 5 min, whereas fractions A02 and A03, were purified under two isocratic methods using 15:85 water/acetonitrile and 70:30 water/ acetonitrile for 5 and 15 min respectively. Fractions A04, A05 and A07 were purified with a linear gradient of water/acetonitrile following the method: 70:30–30:70 over 15 min then 30:70 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. Fraction A06 was purified under an isocratic method using 50:50 water/acetonitrile during 20 min. Fraction A10 was purified with a linear gradient of water/acetonitrile following the method: 60:40 to 52:48 over 2 min, 52:48 to 36:64 over 13 min, 36:64 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. Finally, fractions A08 and A14 were purified with a linear gradient of water/acetonitrile following the method: 80:20 to 20:80 over 15 min then 20:80 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. After HPLC purification, a total of 14 compounds were isolated from phase A (dichloromethane). Compound 22 (Rt = 8.37 min; 0.80 mg) was obtained from fraction A02 (39.10 mg), whereas compounds 6 (Rt = 11.15 min; 0.50 mg) and 12 (Rt = 11.35 min; 1.00 mg) were obtained from purification of A04 (18.70 mg). Compounds 21 (Rt = 7.70 min; 0.40 mg) and 26 (Rt = 7.95 min; 1.00 mg) were obtained from puri- 1175 fication of fraction A05 (61.50 mg), whereas compound 11 (Rt = 10.8 min; 2.30 mg) was obtained from A06 (34.70 mg). Compound 3 (Rt = 9.8 min; 0.40 mg) was isolated after purification of A07 (2.30 mg). Purification of A08 (20.50 mg) yielded a fraction containing a mixture of compounds 9 (Rt = 9.05 min; 0.69 mg) and 10 (Rt = 9.05 min; 0.75 mg). Five different compounds were isolated from fraction A10 (21.90 mg): compound 1 (Rt = 7.60 min; 0.50 mg), a mixture of compounds 7 (Rt = 8.04 min; 1.30 mg), 8 (Rt = 8.8 min; 0.90 mg) and 2 (Rt = 9.3 min; 1.20 mg) and the pure compound 27 (Rt = 12.4 min; 3.20 mg). Analyses of phase A before fractionation (Fig. 2) and the determination of purity of all fractions obtained were performed by HPLC using a linear gradient of water/acetonitrile according to the following method: 10:90– 0:100 over 20 min then 100% acetonitrile for 5 min. Peaks were observed at four wavelengths (214, 254, 280 and 320 nm). Fig. 2 shows the typical RP-HPLC chromatogram of phase B (523 mg), which was also composed of a complex mix of different molecules. Phase B was primarily fractionated by silica gel column chromatography (40–63 lm; 2  50 cm) with a polarity gradient of hexane–ethyl acetate 60:40; 50:50; 40:60; 30:70; 20:80; 10:90 then 100% ethyl acetate and finally 100% methanol. Fifteen fractions were obtained and numbered B01-B15. TLC analyses performed after column chromatography showed that, in the case of this polar phase, more complex mixtures of compounds were present in each obtained fraction. Fractions B01; B03; B06-14 were purified on HPLC using a semi-preparative column while fractions B02, B04, B05 and B15 were not considered for sub-fractionation due to their insufficient mass (<1 mg) and the presence of complex mixtures of compounds as revealed by the TLC analyses. Fraction B01 was purified using a linear gradient following the method: 90:10 to 100% acetonitrile over 20 min and then 100% acetonitrile for 5 min. Fraction B03 was purified using a linear gradient of water/ acetonitrile following the method: 90:10 to 62:38 over 6 min then 62:38 to 56:44 over 9 min, then 56:44 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. Fraction B06 was purified with a linear gradient of water/acetonitrile following the method: 80:20 to 40:60 over 20 min then 40:60 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. Fraction B07 was purified with a linear gradient of water/acetonitrile following the method: 90:10 to 71.5:28.5 over 12.50 min, hold in isocratic mode over 5 min, then 71.5:28.5 to 67.5:32.5 over 3 min, then 67.5:32.5 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. Fraction B08 was purified following the method: 90:10 to 50:50 over 20 min then 50:50 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. Fraction B09 was purified with a linear gradient of water/acetonitrile following the method: 90:10 to 74.2:25.8 over 12.0 min, hold over 10 min, then 74.2:25.8 to 100% acetonitrile over 10 min and then 100% acetonitrile for 5 min. Fraction B10 was purified with a linear gradient of water/ acetonitrile following the method: 90:10 to 65:35 over 30 min then 65:35 to 100% acetonitrile over 5 min and then 100% acetonitrile for 5 min. Fractions B11-B14 were represented by complex mixtures of different molecules and contained very polar compounds; thus they were purified using a linear gradient of water/acetonitrile following the method: 99:1 to 70:30 over 35 min then 100% acetonitrile for 5 min. A total of 20 compounds were isolated from phase B after HPLC fractionation. Some of them (compounds 1–3, 7, 8, 11 and 27) had already been identified in phase A. Compounds 16 (Rt = 17.54 min; 3.40 mg) and 20 (Rt = 18.41 min; 3.30 mg) were obtained from fraction B06 (7.80 mg). Compounds 14 (Rt = 5.36 min; 0.76 mg), 15 (Rt = 10.17 min; 1.99 mg), a mixture of compounds 17 (Rt = 15.13 min; 2.24 mg); 18 (Rt = 15.13 min; 0.83 mg) and 19 (Rt = 15.13 min; 0.32 mg) as well as compound 11 (Rt = 19.08 min; 1.50 mg) were obtained from purification of fraction B07 (36.60 mg). Compound 23 (Rt = 12.3 min; 0.50 mg), 3 (Rt = 17.51 min; 0.70 mg) as well as compound 27 (Rt = 21.21 min; 1176 M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 BS-EAcf (676 mg) suspended in water (10 mg.mL-1) Dichloromethane (2 × 67.6 mL) PHASE A (152.8 mg) Aqueous layer Silica gel column Hexane/Ethyl acetate: 70:30-30:70 (v:v) Ethyl acetate (3 × 67.6 mL) PHASE B (522.95 mg) A00 B03 B06 B07 B08 (16) (20) (14) (15) (17) (18) (19) (11) (23) (3) (27) B09 B10 B11 (28) (25) (4) (5) (1) (8) (2) A02 (22) Silica gel column Hexane/Ethyl acetate: 60:40-10:90 (v:v); 100% ethyl acetate; 100% methanol B01 A01 B12 B13 A03 A04 (6) (12) A05 (21) (26) A06 (11) A07 A08 A10 (3) (9) (10) (1) (7) (2) (8) (27) A14 B14 (13) (24) Fig. 1. Schematic diagram used for the isolation of individual compounds present in the ethyl acetate fraction. Numbers of isolated compounds (represented with black font) correspond to those presented in Fig. 3. 0.7 mg) were isolated from fraction B08 (15.00 mg). Compound 28 (Rt = 3.05 min; 1.38 mg) was obtained from purification of B10 (45.40 mg). Compound 25 (Rt = 11.9 min; 0.60 mg), a mixture of compounds 4 (Rt = 13.65 min; 1.50 mg) and 5 (Rt = 13.65 min; 1.00 mg) as well as compounds 1 (Rt = 13.98 min; 5.50 mg), 8 (Rt = 16.32 min; 1.80 mg) and 2 (Rt = 16.93 min; 1.60 mg) were isolated from B11 (35.60 mg). Purification of B14 (97.80 mg) yielded a mixture of compounds 13 (Rt = 3.1 min; 0.48 mg) and 24 (Rt = 3.1 min; 0.24 mg). Analyses of phase B before fractionation (Fig. 2) and the determination of purity of all fractions obtained were performed by HPLC using a linear gradient of water/acetonitrile as follows: 99:1 to 30:70 over 25 min, then 100% acetonitrile for 5 min. Peaks were observed at four wavelengths (214, 254, 280 and 320 nm). 2.7. Identification of isolated compounds A total of twenty-eight known compounds were identified from BS-EAcf. Considering that compounds were isolated from BS-EAcf, which represents a fraction (20.8% w/w) of the hydrophilic molecules present in BSHWE, the yields of isolated compounds were expressed as a percentage of BS-EAcf (w/w). The structures of these compounds were confirmed by comparing their physical and spectroscopic data (UV, [a], 1H, 13C NMR and MS) with those of corresponding authentic samples or with the values found in the literature. Isolated compounds included five neolignans: cedrusin (1) (Kim et al., 2005), dihydrodehydrodiconiferyl alcohol (2) (Fukuyama, Nakahara, Minami, & Kodama, 1996), 2,3-dihydro3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-(2S,3S)-1,4benzodioxin-6-propanol (3) (Fang, Lee, & Cheng, 1992; Gu, Jing, Pan, Chan, & Yang, 2000) and a mixture of threo (4) (Matsuda & Kikuchi, 1996; Ouyang et al., 2011) and erythro 3-methoxy-8,40 oxyneolignan-30 ,4,7,9,90 -pentol (5) (Fang et al., 1992; Ouyang et al., 2011); seven lignans: a-conidendrin (6) (Davies & Jin, 2003), isolariciresinol (7) (Eklund, Sillanpaa, & Sjoholm, 2002), secoisolariciresinol (8) (Moon, Rahman, Kim, & Kee, 2008), a mixture of 7(R) (9) and 7(S)-hydroxymatairesinol (10) (Fischer, Reynolds, Sharp, & Sherburn, 2004), pinoresinol (11) (Guz & Stermitz, 2000; Moon et al., 2008; Xie, Akao, Hamasaki, Deyama, & Hattori, 2003), epi-pinoresinol (12) (Rahman, Dewick, Jackson, & Lucas, 1990; Swain, Brown, & Bruton, 2004); three phenolic acids: protocatechuic acid (13), vanillic acid (14) and trans-pcoumaric acid (15); one stilbene: trans-resveratrol (16) (FerreFilmon, Delaude, Demonceau, & Noels, 2005; Lee et al., 2001); four flavonoids: a mixture of dihydroquercetin (taxifolin) (17) (Kiehlmann & Li, 1995; Lee et al., 2003; Lundgren & Theander, 1988), pallasiin (18) (Marques-de Oliveira, dos Santos-Humberto, da Silva, Almeida-Rocha, & Goulart-Sant’Ana, 2006; Sakushima, M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 1177 Fig. 2. Typical RP-HPLC chromatogram (k = 214 nm) of phase A (upper pannel) and B (lower pannel) after liquid–liquid partition of the ethyl acetate soluble fraction (BS-EAcf) with dichloromethane (phase A) and ethyl acetate (phase B). Peak numbers correspond to compounds presented in Fig. 3. Coskun, Hisada, & Nishibe, 1983), and (±) epi-taxifolin (19) (Kiehlmann & Li, 1995; Lee et al., 2003; Lundgren & Theander, 1988), mearnsetin (20) (Marques-de Oliveira, dos Santos-Humberto, da Silva, Almeida-Rocha, & Goulart-Sant’Ana, 2006; Rabesa & Voirin, 1979); four other phenolic compounds: dihydroconiferyl alcohol (21), p-vanillin (22), homovanillyl alcohol (23) (Christophoridou & Dais, 2009), orcinol (24); four non-phenolic compounds: 2-[4(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol (25) (Kouno, Yanagida, Shimono, Shintomi, & Yang, 1992), 10-hydroxyverbenone (26) (Yildirim, 2011), 7-oxo-15-hydroxydehydroabietic acid (27) (Yang et al., 2010), levulinic acid (28). 3. Results and discussion 3.1. Polyphenols classes present in the ethyl acetate fraction As can be observed from Table 1, the ethyl acetate fraction, BSEAcf, contained higher total phenol, proanthocyanidins and hydroxycinnamic acid content than the crude aqueous extract of black spruce bark, BSHWE. Other studies have also shown that the ethyl acetate fraction obtained after fractionation of polar crude extracts presents higher phenol contents as determined by the Folin–Ciocalteu method (Joubert, Winterton, Britz, & Gelderblom, 2005). BSHWE is a crude extract composed of a complex mixture of phenolic and non-phenolic hydrophilic molecules. Therefore, the higher phenol and hydroxycinnamic acid contents in BS-EAcf could be explained by the purification and concentration of phenolic compounds throughout the fractionation procedure, starting from the crude aqueous extract. As for the proanthocyanidins (PAs) content, results of the present study differ from those obtained in our previous work in which the content of PAs in BSHWE was higher than that of BS-EAcf (290 vs. 148 mg PAs g 1 extract, respectively) (Diouf et al., 2009). Differences between these results can be explained by the use of different PAs standards for calibration. It is well known that whilst the acid–butanol assay confirms the presence of a polymeric interflavan structure unambiguously, the choice of the different standards can influence the yield of anthocyanidins (Schofield, Mbugua, & Pell, 2001). Results presented here were obtained using the monomer cyanidin chlo- 1178 M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 Table 1 Total phenol, flavonoid, hydroxycinnamic acid and proanthocyanidin contents of hot water extract from Picea mariana bark and its ethyl acetate fraction. Extracts a BS HWE BS-EAcf Total Phen (mg GAE/g) 404 ± 4.04 504 ± 26.20* Total Flav (mg QE/g) * 53.4 ± 1.05 41.7 ± 1.02 Total CinnAc (mg ChAE/g) PAs content (mg CChE/g) 90.3 ± 0.92 113 ± 1.41* 11.8 ± 0.11 19.0 ± 0.55* BSHWE = black spruce extract obtained by hot water extraction; BS-EAcf = ethyl acetate fraction isolated from BSHWE. Total Phen = total phenols content; Total Flav = total flavonoids content; Total CinnAc = total hydroxycinnamic acids content, PAs content = proanthocyanidins content. mg GAE/g = milligrams of gallic acid equivalents (GAE) per gram of dry extract; mg QE/g = milligrams of quercetin equivalents (QE) per gram of dry extract; mg ChAE/ g = milligrams of chlorogenic acid equivalents (ChAE) per gram of dry extract; mg CChE/g = milligrams of cyanidin chloride equivalents (CChE) per gram of dry extract. a Contents of total phenols, flavonoids, hydroxycinnamic acid and proanthocyanidines for BSHWE were previously determined by Garcia-Perez et al., 2010. * p < 0.05 Student’s t test. ride as standard, whereas in our previous study, the purified PAs were isolated directly from the crude black spruce bark aqueous extract. It has been reported that in some cases tannin polymers are incompletely converted by the HCl–ferric ammonium sulphate treatment, into dimers or trimers rather than into monomers, thus leading to underestimation of the content of PAs (Schofield et al., 2001). Concerning the amount of flavonoids, a higher content was determined in BSHWE than in BS-EAcf. That could be explained by the presence of some glycoside-bound flavonoids in the crude extract which were not soluble in ethyl acetate (Ahmadu, Hassan, Abubakar, & Akpulu, 2007). Indeed, compounds such as quercetin glycoside, kaempferol glycoside, dihydroquercetin-30 -O-b-D-glucopyranoside and isorhamnetin-3-O-(600 -O-acetyl)-b-D-glucopyranoside have been found in barks, needles and cones of the Picea spp. (Harris et al., 2008; Pan & Lundgren, 1995). 3.2. Isolation of individual compounds present in the ethyl acetate fraction Very few studies exist to date on the characterisation of polyphenolic compounds in P. mariana (Miller) B.S.P bark. In fact, most of the data are only qualitative, and unequivocal identification, for example, by mass spectrometry or NMR, is lacking. Fractionation of BS-EAcf led to the isolation, quantification and characterisation of 28 known compounds (Fig. 3). On the whole, the five major compounds isolated were cedrusin (1) (0.89% w/w of BS-EAcf) followed by 7-oxo-15-hydroxydehydroabietic acid (27) (0.58% w/w of BS-EAcf), pinoresinol (11) (0.56% w/w of BS-EAcf), trans-resveratrol (16) (0.50% w/w of BS-EAcf) and mearnsetin (20) (0.49% w/w of BS-EAcf). When regrouped in different phenol classes, predominant compounds identified in this fraction were neolignans and lignans (3.57% w/w of BS-EAcf) followed by flavonoids (0.99% w/w of BS-EAcf). 3.2.1. Neolignans Neolignans represent 1.94% (w/w) of BS-EAcf. The predominant neolignan found in this fraction was cedrusin (1) (0.89% w/w) followed by the mixture of threo (4) and erythro 3-methoxy-8,40 oxyneoligna-30 ,4,7,9,90 -pentol (5) (0.48% w/w), dihydrodehydrodiconiferyl alcohol (2) (0.41% w/w) and compound 3 (0.16% w/w). Cedrusin derivatives such as cedrusin-4-O-glucoside, cedrusin-4O-rhamnetin and cedrusin-methyl-4-O-glucoside have been identified in the needles of Norway spruce (Rummukainen, Julkunen-Tiitto, Raisanen, & Lehto, 2007). As to compound 2, it has been identified in the bark of Picea jezoensis (Wada, Yasui, Hitomi, & Tanaka, 2007; Wada, Yasui, Tokuda, & Tanaka, 2009) and in a suspension culture from the seedling leaves of Picea glehnii (Nabeta, Hirata, Ohki, Samaraweera, & Okuyama, 1994). However, to the best of our knowledge, no reports exist concerning the occurrence of compounds 3–5 in the Picea genus. 3.2.2. Lignans Lignans are of great interest in the search for novel agents with antiproliferative, antioxidant and anti-inflammatory properties (Coy, Cuca, & Sefkow, 2009; Di Micco et al., 2011). These compounds represent 1.63% (w/w) of BS-EAcf. The predominant lignan found in this fraction was the pinoresinol (11) (0.56% w/w) followed by secoisolariciresinol (8) (0.40% w/w of BS-EAcf), isolariciresinol (7) (0.24% w/w of BS-EAcf) and epi-pinoresinol (12) (0.15% w/w of BS-EAcf). Additionally, 7(R)-hydroxymatairesinol (9) (0.10% w/w of BS-EAcf), 7(S)-hydroxymatairesinol (10) (0.11% w/w of BS-EAcf) and a-conidendrin (6) (0.07% w/w of BS-EAcf), were identified in this fraction. Lignans have been extensively analysed in some spruce species, mainly in Norway spruce, for a long time (Ekman, 1976; Mattinen, Sjoholm, & Ekman, 1998). Indeed, the knots of this species contain extremely large amounts of these compounds, 6–24% (w/w), with hydroxymatairesinol comprising 65–85% of all lignans (Willfor, Hemming, Reunanen, Eckerman, & Holmbom, 2003). Lignans such as secoisolariciresinol, 7-hydroxymatairesinol, matairesinol, a-conidendrin, pinoresinol and isolariciresinol have been described in Picea abies, Picea glauca and Picea omorika knots and heartwood (Willfor, Nisula, Hemming, Reunanen, & Holmbom, 2004; Willfor et al., 2003). epi-Pinoresinol has been determined by Weinges (1960) from the callus resin of Picea abies as a product of biosynthesis, although it is known that this compound can be formed from pinoresinol in acidic solutions (Lindberg, 1950). To the best of our knowledge, no exhaustive studies exist about the lignan composition in P. mariana bark, but it has been demonstrated that the knots of this wood species contain more lignans than the corresponding stemwood (1–5% vs. 0.2% w/w, respectively) (Willfor et al., 2004). With the exception of isolariciresinol and epi-pinoresinol, lignans such as pinoresinol, secoisolariciresinol, 7-hydroxymatairesinol and a-conidendrin have also been described in black spruce heartwood and knots (Pietarinen, Willfor, Ahotupa, Hemming, & Holmbom, 2006; Willfor et al., 2004). In addition, the presence of other lignans not identified in our study, such as liovil, lariciresinol, matairesinol and cyclolariciresinol have been reported in P. mariana knots and heartwood (Willfor et al., 2004). Although BS-EAcf represents a fraction of the hydrophilic molecules present in BSHWE, our results suggest that pinoresinol could be the dominant lignan in the bark, whereas the two epimers of the 7-hydroxymatairesinol were determined to be the predominant lignans found in knots and heartwood (Willfor et al., 2004). 3.2.3. Phenolic acids Phenolic acids have a potential protective role against oxidative stress and can act as anti-inflammatory agents (Fernandez, Saenz, & Garcia, 1998). These compounds represent 0.47% (w/w) of BS-EAcf. The predominant phenolic acid by far isolated from this fraction was trans-p-coumaric acid (15) (0.29% w/w) followed by vanillic acid (14) (0.11% w/w) and protocatechuic acid (13) (0.07% w/w). The protocatechuic and vanillic acids have been previously identified in the needles of Picea abies (Rummukainen et al., 2007; Soukupova, Cvikrova, Albrechtova, Rock, & Eder, 2000). Furthermore, compound 13 has also been described in the bark of Picea jezoensis (Wada et al., 2007; Wada et al., 2009), whereas compound 15 has been identified in the root bark from Norway 1179 M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 OH HO OH O OH O OH HO OH O O O O HO HO R OH O HO 1 R = OH 2 R = OCH3 3 H O O O O OH OH O HO HO H 4 7R, 8R 5 7S, 8S HO OH OH 6 7 O HO HO H HO O O O H O O OH OH O OH 8 9 7R 10 7S O HO OH O CO2H R2 HO R1 O O 15 HO 13 H CO2H 14 CH3 CO2H O 11 7'S 12 7'R HO O OH OH O OH HO OH OH O R1 H OH H 16 O OH 17 18 19 OH OH O R2 O HO 22 CH3 CHO R1 HO OH R2 R1 OH O R2 H 2R, 3R CH3 2R, 3R H cis R 20 OH 24 21 R = CH2OH 23 R = OH OH O HO O O OH OH HO O OH 25 26 HO2C H 27 O O 28 Fig. 3. Chemical structures of the compounds isolated from the ethyl acetate soluble fraction (BS-EAcf) obtained from the hot water extract of black spruce bark (BSHWE). spruce (Pan & Lundgren, 1995) and in twigs and leaves of Picea neoveitchii (Song et al., 2011). 3.2.4. Stilbenes In this study, the only stilbene found in BS-EAcf was trans-resveratrol (16). This compound constitutes one of the major isolated molecules, representing 0.50% (w/w) of this fraction. In a previous work, it was demonstrated that resveratrol was formed by the partially purified stilbene synthase enzyme from cell culture extracts from Picea excels (Rolfs & Kindl, 1984). Moreover, a recent study suggests that the formation of resveratrol could be the first step for the biosynthesis of other major tetrahydroxystilbenes, astringin and isorhapontin, widely present in spruce bark (Hammerbacher et al., 2011). However, in our study, these compounds and other characteristic stilbene glycosides such as isorhapontigenin and astringenin described in the bark of P. mariana, Picea engelmannii, Picea glauca, Picea rubens, Picea abies, Picea sitchensis and Picea glehnii (Manners & Swan, 1971; Pan & Lundgren, 1995; Pearce, 1996; Shibutani, Samejima, & Doi, 2004) were not found. That is particularly surprising, considering that some of these compounds have also been identified in the ethyl acetate soluble fraction (Shibutani et al., 2004). 1180 M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 trans-Resveratrol displays antioxidant and anti-inflammatory properties (Kalantari & Das, 2010). Previously, in a multicentre double blind clinical study, psoriatic patients treated twice a day for a month with 1% resveratrol ointment showed a marked improvement of their psoriasis compared to the control group (Pellicia, Gianella, & Gianella, 2001). Therefore, trans-resveratrol could be one of the molecules responsible for the in vitro antiinflammatory properties of BS-EAcf on psoriatic keratinocytes, but further investigation should be accomplished to demonstrate its anti-psoriatic properties alone or in combination with other compounds here identified. trans-Resveratrol has been widely studied in grapes and red wines. Even though many factors such as the plant variety, environmental conditions, extraction procedure and solvent used during extraction can influence the quantification of this compound in plant tissues (Roldan, Palacios, Caro, & Perez, 2003; Zhao & Hall, 2008), it reaches about 40.6 lg g 1 in the aqueous extract from Thompson seedless dried grapes (Zhao & Hall, 2008). Other edible and non-edible sources of resveratrol include dark chocolate (0.4 lg g 1) (Counet, Callemien, & Collin, 2006), peanuts (0.03– 0.14 lg g 1) (Sanders, McMichael, & Hendrix, 2000) and the roots of the Polygonum cuspitadum used in ancient Chinese and Japanese herbal medicines (2960–3770 lg g 1) (Vastano et al., 2000). Taking into account the yield obtained through extraction (32.6 g dry bark spruce bark/3.25 g BSHWE/676 mg BS-EAcf), the initial P. mariana dry bark contained at least 104 lg g 1 of trans-resveratrol and could be considered as a new and profitable source of this molecule. In the same way, BS-EAcf contains 503 mg trans-resveratrol/ 100 g of dry extract. When compared with the content of this molecule in other commercial polyphenolic extracts as determined by Counet et al. (2006), one can see that the content of this compound in BS-EAcf, even if present in lower quantities than in Polygonum cuspitadum extract (19719 mg/100 g dry extract), is still higher than in commercial red wine (337 mg/100 g dry extract), red grape skin (60–75 mg/100 g dry extract), white grape skin (63 mg/100 g dry extract), red grape seed (27 mg/100 g dry extract) and white grape seed extracts (25 mg/100 g dry extract). Therefore, the BSEAcf can be regarded as a rich source of resveratrol. That is particularly interesting considering that this compound has a wide range of nutraceutical and phytopharmaceutical properties. 3.2.5. Flavonoids Flavonoids have been investigated in some Picea species with respect to their chemistry, their occurrence in different parts of the trees, and their biological importance. Within Picea, flavonols such as kaempferol and quercetin have been frequently reported in different tissues (Ivanova, Medvedeva, Lutskii, Tyukavkina, & Zelenikina, 1975; Slimestad, Andersen, Francis, Marston, & Hostettmann, 1995; Slimestad, Francis, & Andersen, 1999; Slimestad & Hostettmann, 1996; Song et al., 2011). Some flavonols, glycolysated at the 3-position, such as kaempferol-3-glucoside (glc), quercetin-3-glc and isorhamnetin-3-glc, have been detected in buds and juvenile needles of P. mariana (Slimestad, 2003). However, very few studies have reported the occurrence of these compounds in bark. Flavonoids represent 0.99% (w/w) of BS-EAcf. The major flavonoids identified in this fraction were mearnsetin (20) (0.49% w/w) followed by dihydroflavonols such as dihydroquercetin (taxifolin) (17) (0.33% w/w), pallasiin (18) (0.12% w/w of BSEAcf) and (±) epitaxifolin (19) (0.05% w/w). Recently, mearnsetin was identified for the first time in Picea genus, specifically in the ethyl acetate-soluble fraction (EAcf) of the ethanolic extract obtained from the twigs and leaves of Picea neoveitchii (Song et al., 2011). However, its yield in EAcf was lower (8.69  10 5% w/w) (Song et al., 2011) than that obtained in BS-EAcf. The presence of dihydroquercetin-30 -O-b-D-glucopyranoside has been reported in Picea abies root bark (Pan & Lundgren, 1995). Taxifolin has also been identified in the barks of P. mariana, Picea engelmannii, Picea glauca, Picea rubens (Manners & Swan, 1971) and Picea jezoensis (Wada et al., 2007; Wada et al., 2009). In our previous work, the use of HPLC-DAD techniques allowed the identification of this compound from BS-EAcf (66.7 mg g 1) (Diouf et al., 2009). The high yield previously reported could be explained by difficulties in its separation and by the inherent limitations of the analytical techniques used. In fact, in the present study, compounds 17–19 eluted as a mixture at 15.13 min and only the use of spectroscopic data (1H and 13C NMR, and MS) allowed their accurate characterisation. To our knowledge, this is the first report on the presence of compounds 18 and 19 in P. mariana bark and in Picea genus. It is important to note that even though compound 19 has been identified in other plants such as Anastatica hierochuntica (Nakashima et al., 2010), it can also result from C-2 epimerisation of taxifolin in hot aqueous solutions (Kiehlmann & Li, 1995) and therefore it could be an artifact. 3.2.6. Other phenolic compounds Other phenolic compounds were also identified in BS-EAcf, such as p-vanillin (22) (0.12% w/w), homovanillyl alcohol (23) (0.07% w/ w), dihydroconiferyl alcohol (21) (0.06% w/w of BS-EAcf) and orcinol (24) (0.04% w/w of BS-EAcf). Compound 22 has been found in the wood of Picea koraiensis, Picea ovobata and Picea ajartensis (Leont’eva, Modonova, & Tyukavkina, 1974). Compound 21 is involved in biosynthetic pathway of lignins (Savidge & Forster, 2001) and was identified as a product of lignin degradation in wood of Picea glauca and Picea abies (Arias et al., 2010; Pepper & Lee, 1969). To our knowledge, no reports exist concerning the occurrence of compounds 23 and 24 in Picea species; therefore this is the first report on their occurrence in this genus. 3.2.7. Miscellaneous Although most chemical constituents identified from BS-EAcf were phenolic compounds (84.91% w/w of the total mass of isolated molecules), other non-phenolic molecules were also found in this fraction. Indeed, 7-oxo-15-hydroxydehydroabietic acid (27), a diterpenoid acid, was one of the predominant isolated compounds (0.58% w/w of BS-EAcf). Abietane-type diterpenes, mainly oxidised derivatives, are considered as bioactive molecules (Kinouchi et al., 2000) and their occurrence has been documented in P. mariana heartwood (Conner, Diehl, & Rowe, 1980) and in the stem bark of Picea glehnii (Kinouchi et al., 2000). Other non-phenolic compounds also isolated from BS-EAcf, were levulinic acid (28) (0.20% w/w), 10-hydroxyverbenone (26) (0.15% w/w) and 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol (25) (0.09% w/w of BS-EAcf). Compound 26 is reported to be formed as a result of verbenone biotransformation by some microorganisms (Yildirim, 2011), whereas verbenone has been identified as a product of trans-verbenol oxidation after treatment of Picea abies cells with (R), ( S) and rac-a-pinene (Vanek, Halik, Vankova, & Valterova, 2005). No reports were found about the occurrence of compound 25 in Picea spp. Levulinic acid, a common product of acid transformation of hexose sugars, has been identified as a result of acid hydrolysis of Norway spruce wood (Larsson et al., 1999). 4. Conclusion To the best of our knowledge, this is the first exhaustive report cataloguing the polyphenols in P. mariana bark. Indeed, these results represent an important addition to the information on the phytochemical composition of hydrophilic extractives present in black spruce bark. This study also constitutes the first report describing the presence of the following compounds: 2,3-dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)- M.-E. García-Pérez et al. / Food Chemistry 135 (2012) 1173–1182 (2S,3S)-1,4-benzodioxin-6-propanol, (3), threo and erythro 3-methoxy-8,40 -oxyneolignan-30 ,4,7,9,90 -pentol (4, 5), pallasiin (18), (±) epi-taxifolin (19), homovanillyl alcohol (23), orcinol (24) and,2[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-propanediol (25) in the Picea genus. The isolation and characterisation of these compounds was a difficult task, considering the high number of isomers and the diversity of molecules present in the bark. Neolignans and lignans were the major compounds isolated from the ethyl acetate soluble fraction. Interestingly, the major polyphenols here identified stemmed from the phenylpropanoid biosynthetic pathways, shared with that of lignins. The forest trees are vascular plants characterised by a high lignification of tissues and therefore they represent precious sources of phenylpropanoid molecules displaying numerous bioactivities also found in vegetables and fruits. Indeed, some of the dominant molecules among those isolated from the ethyl acetate fraction of black spruce bark aqueous extract (pinoresinol (11), trans-resveratrol (16), mearnsetin (20) and 7-oxo-15-hydroxydehydroabietic acid (27) possess important antioxidant and anti-inflammatory properties. Furthermore, other minor compounds also identified in this study (isolariciresinol (7), secoisolariciresinol (8), 7(R) and 7(S) hydroxymatairesinol (9, 10), trans-p-coumaric acid (15) and taxifolin (17), are recognised as bioactive polyphenols and could contribute to the anti-inflammatory and antioxidant activity of BS-EAcf, which was determined in our previous studies. From the chemical composition described above, the P. mariana bark and the BS-EAcf could be considered as a new source of bioactive molecules, particularly as an alternative rich source of trans-resveratrol. However, a more accurate quantification of this compound in the bark should be performed, taking into account different solvents, extraction procedures and all fractions that compose BSHWE. Considering the broad spectrum of properties of molecules identified in the extract of black spruce bark, the exploitation of this low-cost and abundant renewable resource can be anticipated for the pharmaceutical or alimentary industries. Indeed, the extraction and purification techniques reported in this study could be used for the production of sufficient quantities of pure compounds to study their potential as antioxidant and anti-inflammatory agents to be used in the formulation of new nutraceutical and/ or pharmaceutical products. Acknowledgments The authors are very grateful to the Natural Science and Engineering Research Council of Canada (NSERC) and to the Canadian Institutes of Health Research (CIHR) for the financial support of this project (research grant to YD, RP and TS). The Natural Science and Engineering Research Council of Canada (NSERC) (research grant to TS, and scholarship to MEGP) and the ‘‘Fonds d’enseignement et de recherche’’ (FER) of the Faculté de Pharmacie, Université Laval, Québec, QC, Canada (scholarship to MEGP) are also acknowledged. RP was recipient of a research fellowship from the «Fonds de la Recherche en Santé du Québec» (FRSQ) of Québec, Canada. The technical support of M. Yves Bedard of the ‘‘Centre de recherche sur le bois, Université Laval’’ is also gratefully acknowledged by the authors. References Ahmadu, A. A., Hassan, H. S., Abubakar, M. U., Akpulu, I. N., et al. (2007). Flavonoid glycosides from Byrsocarpus coccineus leaves. Schum and thonn (connaraceae). 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